Determination of uric acid in blood with uricase

ABSTRACT

A process for quantitively determining uric acid in blood with uricase characterized by making microbe-originated uricase act on uric acid in blood in the presence of a catalase inhibitor and a buffer effective to maintain the pH at 5.5 to 7.0 and measuring the quantity of the generated hydrogen peroxide under a condition of a pH of 4.0 to 7.0 in the presence of an anionic surface active agent, a chromogen developing a color with hydrogen peroxide and a substance having a function of separating free oxygen from hydrogen peroxide and catalysing the oxidation of the chromogen.

United States Patent (191 Kano et al. 1 Jan. 28, 1975 [54] DETERMINATION OF URIC ACID IN 3,367,842 2/1968 Rupe ct al 195/1015 C BLOOD WITH URICASE 3,475,276 /1969 Kano 195/66 R 3,677,903 7/1972 Bittncr 195/1015 R [75] Inventors; Kunio Kano, lharaki; Naohiro Kayama, Takatsuki; Hiroshi Terashima, lbaraki; Yoshitaka Nakagiri, lnuyama. all of Japan [73] Assignees: Ono Pharmaceutical Co., Ltd.; Toyo Boseki Kabushiki Kaisha, both of Osaka, Japan [22] Filed: Nov. 19, 1971 [21] Appl. No.: 200,582

[] Foreign Application Priority Data Nov. 25, 1970 Japan -103966 [52] U.S. Cl. 195/1015 R, 195/66 R, 424/2 [51] lint. Cl C07g 7/02, Cl2k 1/00 [58] Field of Search...l/l03.5 R, 103.5 C, 63 R; H 424/2 [56] References Cited UNITED STATES PATENTS 3,335,069 8/1967 Wachter /66 R OTHER PUBLICATIONS ltaya et al., Agr. Biol. Chem. 31(11): 1256-1264 (1967).

Primary l;".\'amim'r-David M. Naif Attorney, Agent, or Firm-Bicrman & Bicrman [57] ABSTRACT A process for quantitively determining uric acid in blood with uricase characterized by making microbeoriginated uricase act on uric acid in blood in the presence of a catalase inhibitor and a buffer effective to maintain the pH at 5.5 to 7.0 and measuring the quantity of the generated hydrogen peroxide under a condition of a pH of 4.0 to 7.0 in the presence of an anionic surface active agent, a chromogen developing a color with hydrogen peroxide and a substance having a function of separating free oxygen from hydrogen peroxide and catalysing the oxidation of the chromogen.

14 Claims, N0 Drawings DETERMINATION OF URIC ACID IN BLOOD WITH URICASE This invention relates to a process for the quantitative determination of uric acid in blood by using uricase. This invention comprises making uricase act on uric acid at a pH range of 5.5 to 7.0 so the blood may be converted into allantoin and at the same time, generate hydrogen peroxide thus bringing free oxygen decomposed from hydrogen peroxide catalyzed by the peroxidase into contact with a chromogen whereby a color is developed.

The method of enzymatic determination of a substance in which the substance is converted into an intermediate substance by reacting the substance with an enzyme and then the quantity of the intermediate substance is measured by using another enzyme is already known. For example, in the process for the quantitative determination of glucose in blood with glucose oxidase, glucose oxidase is used for the oxidation of glucose as an auxiliary enzyme and hydrogen peroxide produced by the oxidation of glucose is quantitatively determined by its color development by the oxidation of a chromogen using oxygen liberated by the acid of peroxidase. A process for the enzymatic determination of uric acid in blood using animal-originated uricase is already known in Japanese Patent Publication No. 4440/1966 and U.S. Patent Spec. No. 3,349,006. This process is a method of the quantitative determination of hydrogen peroxide generated by making animal uricase act on uric acid under a condition of a pH of 8.5 to 10. However, at this pH range, the generated hydrogen peroxide is so unstable and particularly in blood, is so readily influenced by catalase or reducing substance in the blood serum that it is impossible to make a high precision measurement of uric acid in the blood.

The present inventors developed an accurate, quick and easy process for the quantitative determination of uric acid in blood by applying the principle of such enzymatic analyzing process. Thus, the present invention is as to a process which is characterized by the measurement of hydrogen peroxide generated by making uricase act on uric acid in blood under a condition of a pH of 5.5 to 7.0, and particularly perferable to make microbe-produced uricase act on uric acid under a condition of a pH of 5.7 to 6.5. Usually, in a pH range of below 5.5, the uricase action on uric acid will remarkably decrease. On the other hand in a pH range of above 7.0, a bad influence of reducing substance such as catalase and vitamin C etc. in blood are observed. In the practical use of the present invention, it is preferable to use microbe uricase, particularly preferable yeast-originated uricase, because the optimum pH of the microbe uricase reaction shifts to the relatively acidic side as compared with the animal-originated uricase, therefore the decomposition of hydrogen peroxide by catalase in blood or autodegradation of hydrogen peroxide seems to be surpressed and hydrogen peroxide can be quantitatively caught by the hydrogen peroxide detecting system. Further explanation of the present invention is as follows.

Microbe-produced uricase obtained from yeast is such that its reaction pH is deviated to the acidic side about 1 more than that of animal originated uricase. Additionally it has sufficient activity even at a pH of 5 or 6 at which animal-originated uricase does not act and under this condition. Therefore the quantitative determination of uric acid in blood which was not possible by a known enzymatic process is now made possible. Surprisingly, a significant improvement in accuracy was observed by the process of the'present invention compared to that of known process, particularly in the following point; when uricase is made to act on uric acid near the pH of 8.0 to 9.0 hydrogen peroxide produced thereby will be so quickly decomposed by the catalase present in the blood serum, or so readily autodegradated by hydroxy. ions and will be so likely to be influenced by reducing substance (such as vitamin C. glutathion, cysteine and SH protein) in the blood serum that the quantitatively determined value of uric acid is lower than its true value and, thus, it will not be able to be utilized pratically.

The above mentioned defects have been all eliminated by the process of the present invention. As shown in the table, when compared with the value measured by a standard quantitative determination method of uric acid (uricametric UV method) wherein the decrease of the absorption of 293 mu or uric acid is measured by using uricase, a measured value of uric acid by using a hydrogen peroxide generating system under the pH range used in the conventional process even if a catalase inhibiting agent (for example, sodium azide) is used to prevent the decomposition of hydrogen peroxide, there is a greater loss of hydrogen peroxide than the value obtained by using the hydrogen peroxide 'generating system of the present invention.

Fresh blood serum (within 24 hours after the blood was taken) 7r Preserved blood serum (in 3 weeks after the blood was taken) Uricametric UV process (293 mp. absorption measurement) Known process (Hydrogen peroxide generating system at a pH of 8.2 to 9.2)

Process of the present invention (Hydrogen peroxide generating system at a pH of 6.0)

Particularly with fresh blood serum, in the system of known process (shown by Japanese Patent Publication No. 4440/1966 or U.S. Pat. No. 3,349,006), the measured value was 55 compared with the value, 100 obtained by the uricametric UV process, and it evidently shows us how large the loss of hydrogen peroxide is and also shows that the practical use of the known process is thought to be impossible, whereas. in the process of the present invention, the measured value is 97 and the practical use of the process is possible. In fresh blood serum, the effects of reducing substance such as vitamin C, glutathion, cystein and SH protein, and catalase which consume hydrogen peroxide are strong, whereas, in preserved blood serum, its influence is little.

The present inventors have clarified how important the effect of pH regulation is for eliminating these influences.

It is absolutely necessary to know the true value of uric acid in blood. By the process of the present invention, which is useful, quick and easy for the quantitative measurement of uric acid in blood with uricase in a clinical diagnosis, it becomes possible to practice a diagnostic testing method. As the buffer to be used to keep the pH under a specific condition for making uricase act on uric acid in blood in the process of the present invention, any buffer which works at a pH range of 5.5 to 7.0 can be used. Generally, a phosphate buffer. citrate buffer and acetate buffer are used in the present invention.

In the process of the present invention, the generated hydrogen peroxide can be measured by any method. Howover, usually it is measured by developing a color of a chromogen with hydrogen peroxide in the presence of a substance, for example peroxidase, which catalyzed the reaction of chromogen with free oxygen produced by decomposition of hydrogen peroxide and then colorimetrically determining it. For the above mentioned chromogen, one can list o-tolidine, 2,7- diaminofluorene, dimethyl-p-phenylenediamine, diethyl-p-phenylenediamine, o-dianisidine and oaminophenol. It is preferable to develop a color by contacting hydrogen peroxide with said chromogen in a buffer solution ofa pH of 4 to 7, most preferably a pH of 4 to 5.5.

In quantitatively determining uric acid in blood by the process of the present invention, as a hydrogen peroxide decomposing enzyme, catalase, is contained in the blood, it is preferable to add a catalase inhibitor in order to prevent the decomposition of the produced hydrogen peroxide. As such catalase inhibitor, for example, sodium azide, potassium cyanide and sodium cyanide can be enumerated. Sodium azide among them is particularly preferable. However, in case such catalase inhibitor as sodium azide is present, when a color is developed in a chromogen with hydrogen peroxide at a pH of 4 to 7 and colorimetrically determined, the fading of the developed chromogen color will be so remarkable that it will be difficult to judge the color development and substantially no color will be developed in the chromogen at a pH of about 7 or higher. In order to prevent such fading, in the process of the present invention, it is preferable to carry out the detection of hydrogen peroxide with chromogen in the presence of an anionic surface active agent. When an anionic surface active agent is added, the color development of said chromogen will become stable, no fading will occur for more than 10 minutes after the color development and it will be possible to accurately measure uric acid. The kind of such anionic surface active agent is not particularly limited and any kind of anionic surface active agent can be used. However, it is particularly preferable to use salts of higher alcohol, sulfates, alkylbenzenesulfonates, alkylnaphthalenesulfonates, alkyl phosphates and dialkyl sulfosuccinates. In case the chromogen is made to develop a color in a solution, it will be preferable to make the concentration of said added anionic'surface active agent more than 0.001 In case the anionic surface active agent is added to a chromogen-containing reagent solution for preparing testing paper for detecting hydrogen peroxide, it will be preferable to add it so that is concentration may be more than 0.01

Further, in case an anionic surface active agent is added to a chromogen as mentioned above, the chromogen oxidated by the produced hydrogen peroxide will become insoluble, cohere and precipitate. In order to prevent such cohesion and precipitation. it is preferable to add such dispersing agent as a polyethylene glycol. polyvinyl alcohol or polyvinyl pyrrolidone. Such dispersing agent has an effect as of a color increasing agent simultaneously with an effect as of a pigment dispersing agent.

In case hydrogen peroxide testing paper is to be used to detect produced hydrogen peroxide in the process of the present invention (see Example 3). the chromogen color can be made easy to judge with the naked eye by adding some pigment such as erythrosin or auramine, etc. so that the tone may be varied.

The process of the present invention can be worked in the presence of not only ethylenediaminetetracetate as a stabilizer for uricase but also any other suitable stabilizers.

The process of the present invention can be applied to blood in general and is particularly useful to the quantitative determination of uric acid in blood serum. However, in the case of measuring uric acid in blood serum containing a high concentration of vitamin C, in order to eliminate the influence on the measured value of uric acid, it is desirable to add a proper amount of copper ions, for example, a small amount of copper sulfate into the blood srum in the process of the present invention. No bad influence of the coexistence of copper ions on the process of the present invention has been recognized.

The process of the present invention practically comprises making a hydrogen peroxide generating system containing uricase, a catalase inhibitor and a buffer effective to maintain the pH at 5.5 to 7.0, act on uric acid, and then detecting the hydrogen peroxide generated with a hydrogen peroxide detecting system containing a chromogen, a substance having a function of catalyzing the oxidation of the chromogen and an anionic surface active agent. However, the generation and detection of hydrogen peroxide can be carried out simultaneously in one system in the present invention.

The present invention shall be explained more particularly with the following examples in which one unit of the uricase activity is defined to be the quantity of the enzyme to catalyze uric acid so as to be converted to allantoin at a rate of one micromol per minute at a temperature of 25C. and one unit of the peroxidase activity is defined to be the quantity of the enzyme having a capacity of forming 1 mg. of purpurogallin per 20 seconds at a pH of 6.0 at 20C.

EXAMPLE 1 The following formulation was mixed to obtain a hydrogen peroxide generating system composition:

lM-sodium phosphate buffer solution (pH 6.0) 1 ml. SOmM-sodium azide 1 ml. Uricase (yeast-originated) (l unit/ml.) l ml.

After the completion of the reaction, the following formulation was added thereto as a hydrogen peroxide detecting system:

Peroxidase (l unit/ml.) l O-dianisidine (l mg./ml.) 0.

They were made to react at the room temperature for 5 minutes. The reaction system was colorimetrically quantitatively determined with a standard uric acid sample solution (of 10.0 mg./dl. of uric acid) having reacted to develop a color under the same condition and obtained a favorable result in which the quantity of uric acid in blood was 10.2 mg./dl.

EXAMPLE 2 Filter paper was impregnated with a solution prepared to be of a total amount of 100 ml by adding Peroxidase 190 purpurogallin units/mg.) 0. O-tolidine hydrochloride Citric anhydride 1. Sodium citrate 6 Polyvinyl pyrrolidone 2. Sodium laurylbenzene sulfonate 0. Erythrosine 0 Alcohol 30 to distilled water and was dried at the room temperature to prepare hydrogen peroxide testing paper. When the thus obtained hydrogen peroxide testing paper was dipped in a solution containing hydrogen peroxide in a concentration range of 2 to 40 ppm., with the increase of the concentration of hydrogen peroxide, a clear color was developed in a range of blue through purple from reddish purple. This color did not change for more than 10 minutes and was stable.

Then 0.1 ml. of lM-phosphate buffer solution (pH 6.0, 0.1 ml. of 0.05M-sodium azide and 0.1 ml. of uricase solution (10 units/ml.) was added to 0.2 ml. of blood serum of each of different concentrations of uric acid, and left at room temperature for 5 minutes and was tested with the above mentioned hydrogen peroxide testing paper. As a result, there were developed reddish purple at a concentration or uric acid of 2 to 5 mg./dl., purple near mg./dl. and clear blue at to mg./dl. These colors did not fade or change for more than 10 minutes. A quantitative determination of high precision was made possible by the use of a standard testing paper color chart. On the other hand, when the tests were made under the same conditions as are mentioned above except using a lM-borate buffer solution (pH 8.5) instead of the lM-phosphate buffer solution (pH 6.0) in the above mentioned serum/uricase solution, reddish purple was shown at a uric acid concentration in a wide range of 2 to 15 mg./dl., the developed shade changed little with the uric acid concentration and it was difficult to judge the concentration high in the precision. When uric acid in blood serum was measured in a borate buffer solution of a pH of 8.5 by using testing paper prepared by impregnating filter paper with a solution of exactly the same composition as is mentioned above except containing no sodium laurylbenzenesulfonate in the preparation of the above mentioned hydrogen peroxide testing paper, the color development was so weak and the fading was so severe that it was impossible to judge the concentration of uric acid.

EXAMPLE 3 lM-sodium citrate buffer solution (pH 5.8) 0.1 ml. Sodium azide (50 mM) 0.1 ml. Uricase (Yeast-originated) 0.05 ml. Ethylenediaminetetracetic acid (1 mM) 0.05 ml.

were mixed together and this composition with the addition of 0.2 ml. of blood serum was made to react at the room temperature for 5 minutes. After the completion of the reaction, when the hydrogen peroxide produced in the reaction solution was tested with the hydrogen peroxide testing paper prepared in Example 2. said testing paper showed a clear reddish purple color development and, when it was compared with a standard testing paper color chart prepared with uric acid of a standard concentration, there was obtained a result that the content of uric acid was 7.0 mg./dl. This value well coincided with the value of7.3 mg./dl. obtained by measuring uric acid. in the same serum sample by an uricametric ultraviolet ray absorption method.

EXAMPLE 4 0.2 ml. of blood serum containing vitamin C of a high concentration with the addition of 0.1 ml. of 10 M copper sulfate was left at the room temperature for 10 minutes and was then made to react at the room for 10 minutes with the addition of a mixed solution of the below mentioned composition:

1 M-sodium phosphate buffer (pH 6.8) 0.1 ml. Sodium azide mM) 0.05 ml. Uricase (yeast-originated) (12 units/ml.) 0.05 ml.

After the completion of the reaction, when the produced hydrogen peroxide was tested with the hydrogen peroxide testing paper prepared in Example 2, there was obtained a result that the uric acid content was 12 mg./dl. This well coincided with the value of 12.5 mg./dl. measured by an ultraviolet ray absorption method. The bad influence by vitamin C had been evidently removed.

What is claimed is:

1. A process for quantitatively determining uric acid in blood with a yeast originated uricase which comprises reacting the uricase with the uric acid in blood at a pH of 5.7-6.5 to generate hydrogen peroxide and measuring the generated hydrogen peroxide.

2. The process as recited in claim 1 wherein a buffer which maintains a pH range of 57-65 is added to the blood.

3. The process as recited in claim 2 wherein the buffer is a phosphate buffer, a citrate buffer or an acetate buffer.

41. The process as recited in claim 1 wherein the hydrogen peroxide is measured by contacting the hydrogen peroxide with a chromogen whereby a color is produced.

5. The process as recited in claim 4 wherein the chromogen is o-tolidine. 2,7-diaminofluorene. dimethyl-pphenylenediamine, diethyl-p-phenylenediamine, odianididine, or o-aminophenol.

6. The process as recited in claim 4 wherein the hy- 11. The process as recited in claim 10 wherein the drogen peroxide is contacted with the chromogen in a di per ing agent is polyethylene glycol, polyvinyl alcobuffer solution having a pl'l of 4- 7. holy of polyvinyl pyrrolidone g s as melted m clam 6 wherein the PH 5 12. The process as recited in claim 1 wherein copper is ions are added to the blood.

13. The process as recited in claim 4 wherein the hy- 9. The process as recited in claim 8 wherein the catadrogen peroxlde contacted the chromogen m lase inhibitor is sodium azide, potassium cyanide, or sothe Presence of a f 'oxldafion catalystdi id l0 14. The process as recited in claim 13 wherein the 10. The process as recited in claim 4 wherein a disly t iS peroxidase. persing agent is added to the chromogen.

8. The process as recited in claim 1 wherein a catalase inhibitor is added to the blood. 

2. The process as recited in claim 1 wherein a buffer which maintains a pH range of 5.7-6.5 is added to the blood.
 3. The process as recited in claim 2 wherein the buffer is a phosphate buffer, a citrate buffer or an acetate buffer.
 4. The process as recited in claim 1 wherein the hydrogen peroxide is measured by contacting the hydrogen peroxide with a chromogen whereby a color is produced.
 5. The process as recited in claim 4 wherein the chromogen is o-tolidine, 2,7-diaminofluorene, dimethyl-p-phenylenediamine, diethyl-p-phenylenediamine, o-dianididine, or o-aminophenol.
 6. The process as recited in claim 4 wherein the hydrogen peroxide is contacted with the chromogen in a buffer solution having a pH of 4-7.
 7. The process as recited in claim 6 wherein the pH is 4 - 4.5.
 8. The process as recited in claim 1 wherein a catalase inhibitor is added to the blood.
 9. The process as recited in claim 8 wherein the catalase inhibitor is sodium azide, potassium cyanide, or sodium cyanide.
 10. The process as recited in claim 4 wherein a dispersing agent is added to the chromogen.
 11. The process as recited in claim 10 wherein the dispersing agent is polyethylene glycol, polyvinyl alcohol, or polyvinyl pyrrolidone.
 12. The process as recited in claim 1 wherein copper ions are added to the blood.
 13. The process as recited in claim 4 wherein the hydrogen peroxide is contacted with the chromogen in the presence of a chromogen oxidation catalyst.
 14. The process as recited in claim 13 wherein the catalyst is peroxidase. 